Inhibitors of Bcl-2

ABSTRACT

Compounds and pharmaceutical compositions thereof for inducing apoptosis in a cell expressing Bcl-2 and IP 3 R and their use in a method for treating neoplastic disorders in a subject.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.62/291,915, filed Feb. 5, 2016, the subject matter of which isincorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. CA085804awarded by The National Institutes of Health. The United Statesgovernment has certain rights in the invention.

TECHNICAL FIELD

This application relates generally to compounds that can be used toinhibit the interaction of Bcl-2 to the inositol 1,4,5-triphosphatereceptor (IP₃R). This application also relates to pharmaceuticalcompositions containing these compounds and methods of using thecompounds for the treatment of Bcl-2 associated diseases and disorders.

BACKGROUND

Apoptosis is an important process in the development of cells and inmaintaining the proper number of cells in the body. Candidates forapoptosis include cells that may be a danger to an organism, such ascells with damaged DNA or cells growing at improper rates. However,apoptosis is also applied to normal cells that have simply becomeobsolete as organisms grow and develop.

Bcl-2 protein is known to inhibit apoptotic cell death. Bcl-2 proteinserves as a check on apoptosis allowing healthy and useful cells tosurvive. Anti-apoptotic molecules, such as Bcl-2 are often overexpressedin cancer cells and their inhibition is an attractive target forselective killing of tumor cells via induction of apoptosis. Bcl-2overexpression and/or activation has been correlated with resistance tochemotherapy, to radiotherapy and to development of hormone-resistanttumors. Inhibition of apoptosis by Bcl-2 contributes to cancer byinhibiting cell death. Thus, inhibiting Bcl-2 activity in cancer cellscan reduce chemotherapeutic resistance and increase the killing ofcancer cells.

The Bcl-2 gene was discovered as the translocated locus in a B-cellleukemia. Bcl-2 contains a single transmembrane domain and is localizedwithin a cell to the outer mitochondrial, nuclear, and endoplasmicreticulum membranes. Bcl-2 was first isolated as a breakpointrearrangement in human follicular lymphomas. In humans, most follicularB-cell lymphomas contain a chromosomal translocation that moves the genefor Bcl-2 from its normal location to a position within the genes forimmunoglobulins. In this new location, higher quantities of Bcl-2 areproduced. Since Bcl-2 is a potent pro-survival protein, it shields thecancer cells from apoptotic instruction.

The effector molecules in the apoptotic pathway are a family of enzymesknown as the caspases. The Bcl-2 protein suppresses apoptosis bypreventing the activation of the caspases that carry out the process.Caspase enzymes are cystein proteases that selectively cleave proteinsat sites just C-terminal to aspartate residues. These proteases havespecific intracellular targets such as proteins of the nuclear laminaand cytoskeleton. The cleavage of these substrates leads to the demiseof a cell.

The inositol 1,4,5-triphosphate (IP₃) messenger molecule is watersoluble, and can diffuse within the cytosol carrying an activated Gprotein signal from the cell surface to the endoplasmic reticulum (ER)surface. IP₃ binds to an IP₃R and induces opening of the channelallowing Ca²⁺ ions to exit from the ER into the cytosol. The releasedcalcium then triggers a mass exodus of cytochrome c from allmitochondria in the cell, thus activating the caspase and nucleaseenzymes that finalize the apoptotic process.

It has previously been shown that Bcl-2 interacts with the inositol1,4,5-triphosphate receptor (IP₃R) and inhibits IP₃-mediated Ca²⁺release from the ER, thereby inhibiting anti-CD3 induced apoptosis inimmature T cells (JCB 166:193-203, 2004; JCB 172: 127-137, 2006). IP₃Rhave a broad tissue distribution and are mostly found in the cellintegrated into the endoplasmic reticulum. The IP₃R is a large sixtransmembrane ligand gated ion channel, which mainly transmits calciumions and thereby facilitates triggers apoptosis.

SUMMARY

This application relates to compounds and pharmaceutical compositionsthereof that inhibit binding of Bcl-2 to IP₃ receptors (IP₃R) of cellsthat express IP₃R and Bcl-2.

In one aspect, the application provides a method of inducing apoptosisin a cell expressing Bcl-2 and IP₃R. The method includes administeringto the cell a therapeutically effective amount of a compound thatinhibits binding of Bcl-2 to IP₃ receptors (IP₃R) of cells that expressIP₃R and Bcl-2.

In some embodiments, the compound is derived from the Bcl-2 bindingdomain of IP³R. The Bcl-2 binding domain of IP₃R can include the BH4binding domain of IP₃R. In some aspects, the compound can include aBIRD-2 (Bcl-2-IP₃R interaction Disrupter-2) mimetic agent. In someaspects, the compound can include a non-peptide BIRD-2 mimetic agent. Incertain aspects, the compound can reverse the interaction of Bcl-2 withIP₃R of cells that express Bcl-2 and IP₃R.

The method can further include administering a second agent to the cellthat inhibits binding of Bcl-2 to BH3 pro-apoptotic proteins and incombination with a compound that inhibits binding of Bcl-2 to IP₃R,induces synergistic cytotoxicity of cells that express Bcl-2 and IP₃R.The second agent can include at least one of a chromene, a thiazolidine,a benzenesulfonyl, a benzenesulfonamide, an antimycin, adibenzodiazocine, a terphenyl, an indole, gossypol, apogossypol, anepigallocatechingallate, or a theaflavin. The second agent can alsoincludeN-(4-(4-(4′-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl)-benzoyl)-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide,ABT-737, ABT-263, and ABT-199.

The application further relates to a method of treating a neoplasticdisorder, such as chronic lymphocytic leukemia or multiple myeloma, in asubject. The method includes administering to neoplastic cells of thesubject expressing IP₃R and Bcl-2 a therapeutically effective amount ofa compound that inhibits binding of Bcl-2 to IP₃ receptors (IP₃R) ofcells that express IP₃R and Bcl-2.

In some embodiments, the compound is derived from the Bcl-2 bindingdomain of IP₃R. The Bcl-2 binding domain of IP₃R can include the BH4binding domain of IP₃R. In some aspects, the compound can include aBIRD-2 (Bcl-2-IP₃R interaction Disrupter-2) mimetic agent. In someaspects, the compound can include a non-peptide BIRD-2 mimetic agent. Incertain aspects, the compound can reverse the interaction of Bcl-2 withIP₃R of cells that express Bcl-2 and IP₃R.

The method can further include administering a second agent to the cellthat inhibits binding of Bcl-2 to BH3 pro-apoptotic proteins incombination with a compound that inhibits binding of Bcl-2 to IP₃R,induces synergistic cytotoxicity of cells that express Bcl-2 and IP₃R.The second agent can include at least one of a chromene, a thiazolidine,a benzenesulfonyl, a benzenesulfonamide, an antimycin, adibenzodiazocine, a terphenyl, an indole, gossypol, apogossypol, anepigallocatechingallate, or a theaflavin. The second agent can alsoincludeN-(4-(4-(4′-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl)-benzoyl)-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide,ABT-737, ABT-263, and ABT-199.

In some aspects the neoplastic disorder includes a Bcl-2 associatedcancer. The Bcl-2 associated cancer can be selected from the groupconsisting of chronic lymphocytic leukemia (CLL), follicular lymphoma,diffuse large B-cell lymphoma, and multiple myeloma (MM).

BRIEF DESCRIPTION OF DRAWINGS

Further features of the present invention will become apparent to thoseskilled in the art to which the present invention relates from readingthe following description of the invention with reference to theaccompanying drawings.

FIG. 1 illustrates a graphic showing the Bcl-2 family. Bcl-2 has fourBcl-2 homology (BH) domains, whereas pro-apoptotic family members lack aBH4 domain. In Bcl-2, BH1-3 form a hydrophobic groove that binds andinhibits full length (e.g., Bax, Bak) and BH3-only (e.g., Bim, Bad)family members. The BH4 domain of Bcl-2 binds to IP₃Rs.

FIG. 2 illustrates a graphic showing that BIRD-2 targets Bcl-2-IP₃Rinteraction. BIRD-2 is a synthetic peptide derived from IP₃R domain 3where Bcl-2 binds. It functions as a decoy peptide, inhibitingBcl-2-IP3R interaction and inducing apoptosis in Bcl-2-positive cancercells.

FIG. 3 illustrates a graphic showing that Bcl-2 inhibits apoptosis intwo distinct ways. (Left) The BH4 domain of Bcl-2 binds to the IP₃R,preventing Ca²⁺ elevations that induce apoptosis. BIRD-2 inhibits thisinteraction, inducing apoptosis by releasing high levels of Ca²⁺ fromthe ER. (Right) BH1-3 domains of Bcl-2 bind BH3-only proteins like BIM,preventing Bim from activating Bax/Bak and inducing apoptosis.BH3-mimetics like ABT-263 and ABT-199 block this interaction, inducingapoptosis.

FIG. 4 is a graph illustrating BIRD-2/ABT-263 synergism. H2171 Smallcell lung cancer (SCLC) cells treated from 24 hr with 5 μM BIB1 and 39nM ABT-263 alone or together.

FIG. 5 illustrates a graphic showing a heat map summarizing HTS resultsidentifying 4 lead compounds that kill Bcl-2-positive cancer cell lines,primary CLL cells and elevate Ca²⁺ in Bcl-2 positive Jurkat cells.

FIG. 6 illustrates charts showing that compound 854697 works on-target,inducing IP₃R-mediated Ca²⁺ elevation. Pretreatment of Jurkat cells for30 min with the phospholipase c inhibitor U73122 to block IP₃ synthesisblocks 854697-induced Ca²⁺ elevation, consistent with hypothesis that854697 disrupts Bcl-2-IP³R interaction, releasing Ca²⁺ from the ER lumeninto the cytoplasm where it is detected by digital imaging.

FIG. 7 is a graph illustrating that 854697 induces Ca²⁺ elevation inprimary human CLL cells. Shown is a trace averaging Ca²⁺ elevationsinduced in 80 CLL cells, consistent with inhibition of Bcl-2-IP₃Rinteraction by 854697.

FIG. 8 is a graph illustrating surface plasmon resonance (SPR) resultsindicating four compounds that bind to the BH4 domain of Bcl-2. Doseresponse testing indicates a Kd of 5.7 μM for 854697 and 24 μM for96505.

FIG. 9 is a graph illustrating surface plasmon resonance (SPR) resultsindicating that 854697 binds to the BH4 domain of Bcl-2 with K_(d) 5.71μM.

FIG. 10 is a graph illustrating the synergistic effect between 854697and the BH3-mimetic ABT-263. KMS-12-BM cells, which express Bcl-2, weretreated as shown with combinations of these agents at fixedconcentration ratios, demonstrating synergy (Cl<1) in inducing celldeath.

DETAILED DESCRIPTION

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisapplication belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “comprise,” “comprising,” “include,” “including,” “have,” and“having” are used in the inclusive, open sense, meaning that additionalelements may be included. The terms “such as”, “e.g.”, as used hereinare non-limiting and are for illustrative purposes only. “Including” and“including but not limited to” are used interchangeably.

The term “or” as used herein should be understood to mean “and/or”,unless the context clearly indicates otherwise.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length. In oneembodiment, the term “about” or “approximately” refers a range ofquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%,±2%, or ±1% about a reference quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length.

It will be noted that the structure of some of the compounds of theapplication include asymmetric (chiral) carbon or sulfur atoms. It is tobe understood accordingly that the isomers arising from such asymmetryare included herein, unless indicated otherwise. Such isomers can beobtained in substantially pure form by classical separation techniquesand by stereochemically controlled synthesis. The compounds of thisapplication may exist in stereoisomeric form, therefore can be producedas individual stereoisomers or as mixtures.

The term “isomerism” means compounds that have identical molecularformulae but that differ in the nature or the sequence of bonding oftheir atoms or in the arrangement of their atoms in space. Isomers thatdiffer in the arrangement of their atoms in space are termed“stereoisomers”. Stereoisomers that are not mirror images of one anotherare termed “diastereoisomers”, and stereoisomers that arenon-superimposable mirror images are termed “enantiomers”, or sometimesoptical isomers. A carbon atom bonded to four nonidentical substituentsis termed a “chiral center” whereas a sulfur bound to three or fourdifferent substitutents, e.g., sulfoxides or sulfinimides, is likewisetermed a “chiral center”.

The term “chiral isomer” means a compound with at least one chiralcenter. It has two enantiomeric forms of opposite chirality and mayexist either as an individual enantiomer or as a mixture of enantiomers.A mixture containing equal amounts of individual enantiomeric forms ofopposite chirality is termed a “racemic mixture”. A compound that hasmore than one chiral center has 2n−1 enantiomeric pairs, where n is thenumber of chiral centers. Compounds with more than one chiral center mayexist as either an individual diastereomer or as a mixture ofdiastereomers, termed a “diastereomeric mixture”. When one chiral centeris present, a stereoisomer may be characterized by the absoluteconfiguration (R or S) of that chiral center. Alternatively, when one ormore chiral centers are present, a stereoisomer may be characterized as(+) or (−). Absolute configuration refers to the arrangement in space ofthe substituents attached to the chiral center. The substituentsattached to the chiral center under consideration are ranked inaccordance with the Sequence Rule of Cahn, Ingold and Prelog. (Cahn etal, Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al.,Angew. Chem. 1966, 78, 413; Cahn and Ingold, J Chem. Soc. 1951 (London),612; Cahn et al., Experientia 1956, 12, 81; Cahn, J., Chem. Educ. 1964,41, 116).

The term “geometric Isomers” means the diastereomers that owe theirexistence to hindered rotation about double bonds. These configurationsare differentiated in their names by the prefixes cis and trans, or Zand E, which indicate that the groups are on the same or opposite sideof the double bond in the molecule according to the Cahn-Ingold-Prelogrules. Further, the structures and other compounds discussed in thisapplication include all atropic isomers thereof.

The term “atropic isomers” are a type of stereoisomer in which the atomsof two isomers are arranged differently in space. Atropic isomers owetheir existence to a restricted rotation caused by hindrance of rotationof large groups about a central bond. Such atropic isomers typicallyexist as a mixture, however as a result of recent advances inchromatography techniques, it has been possible to separate mixtures oftwo atropic isomers in select cases.

The terms “crystal polymorphs” or “polymorphs” or “crystal forms” meanscrystal structures in which a compound (or salt or solvate thereof) cancrystallize in different crystal packing arrangements, all of which havethe same elemental composition. Different crystal forms usually havedifferent X-ray diffraction patterns, infrared spectral, melting points,density hardness, crystal shape, optical and electrical properties,stability and solubility. Recrystallization solvent, rate ofcrystallization, storage temperature, and other factors may cause onecrystal form to dominate. Crystal polymorphs of the compounds can beprepared by crystallization under different conditions.

The term “derivative” refers to compounds that have a common corestructure, and are substituted with various groups as described herein.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres includeacyl sulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g.,Patani and LaVoie, Chem. Rev. 96, 3147-3176 (1996).

The phrases “parenteral administration” and “administered parenterally”are art-recognized terms, and include modes of administration other thanenteral and topical administration, such as injections, and include,without limitation, intravenous, intramuscular, intrapleural,intravascular, intrapericardial, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The term “treating” is art-recognized and includes inhibiting a disease,disorder or condition in a subject, e.g., impeding its progress; andrelieving the disease, disorder or condition, e.g., causing regressionof the disease, disorder and/or condition. Treating the disease orcondition includes ameliorating at least one symptom of the particulardisease or condition, even if the underlying pathophysiology is notaffected.

The term “preventing” is art-recognized and includes stopping a disease,disorder or condition from occurring in a subject, which may bepredisposed to the disease, disorder and/or condition but has not yetbeen diagnosed as having it. Preventing a condition related to a diseaseincludes stopping the condition from occurring after the disease hasbeen diagnosed but before the condition has been diagnosed.

The term “pharmaceutical composition” refers to a formulation containingthe disclosed compounds in a form suitable for administration to asubject. In a preferred embodiment, the pharmaceutical composition is inbulk or in unit dosage form. The unit dosage form is any of a variety offorms, including, for example, a capsule, an IV bag, a tablet, a singlepump on an aerosol inhaler, or a vial. The quantity of active ingredient(e.g., a formulation of the disclosed compound or salts thereof) in aunit dose of composition is an effective amount and is varied accordingto the particular treatment involved. One skilled in the art willappreciate that it is sometimes necessary to make routine variations tothe dosage depending on the age and condition of the patient. The dosagewill also depend on the route of administration. A variety of routes arecontemplated, including oral, pulmonary, rectal, parenteral,transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal,intranasal, inhalational, and the like. Dosage forms for the topical ortransdermal administration of a compound described herein includespowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, nebulized compounds, and inhalants. In a preferred embodiment,the active compound is mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants that are required.

The term “flash dose” refers to compound formulations that are rapidlydispersing dosage forms.

The term “immediate release” is defined as a release of compound from adosage form in a relatively brief period of time, generally up to about60 minutes. The term “modified release” is defined to include delayedrelease, extended release, and pulsed release. The term “pulsed release”is defined as a series of releases of drug from a dosage form. The term“sustained release” or “extended release” is defined as continuousrelease of a compound from a dosage form over a prolonged period.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, polymers and othermaterials and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” is art-recognized, andincludes, for example, pharmaceutically acceptable materials,compositions or vehicles, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting any subject composition from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof a subject composition and not injurious to the patient. In certainembodiments, a pharmaceutically acceptable carrier is non-pyrogenic.Some examples of materials which may serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

The compounds of the application are capable of further forming salts.All of these forms are also contemplated herein.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. For example, the saltcan be an acid addition salt. One embodiment of an acid addition salt isa hydrochloride salt. The pharmaceutically acceptable salts can besynthesized from a parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrilebeing preferred. Lists of salts are found in Remington's PharmaceuticalSciences, 18th ed. (Mack Publishing Company, 1990).

The compounds described herein can also be prepared as esters, forexample pharmaceutically acceptable esters. For example, a carboxylicacid function group in a compound can be converted to its correspondingester, e.g., a methyl, ethyl, or other ester. Also, an alcohol group ina compound can be converted to its corresponding ester, e.g., anacetate, propionate, or other ester.

The compounds described herein can also be prepared as prodrugs, forexample pharmaceutically acceptable prodrugs. The terms “pro-drug” and“prodrug” are used interchangeably herein and refer to any compound,which releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.) the compounds can bedelivered in prodrug form. Thus, the compounds described herein areintended to cover prodrugs of the presently claimed compounds, methodsof delivering the same and compositions containing the same. “Prodrugs”are intended to include any covalently bonded carriers that release anactive parent drug in vivo when such prodrug is administered to asubject. Prodrugs are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds wherein a hydroxy, amino, sulfhydryl, carboxy, orcarbonyl group is bonded to any group that may be cleaved in vivo toform a free hydroxyl, free amino, free sulfhydryl, free carboxy or freecarbonyl group, respectively. Prodrugs can also include a precursor(forerunner) of a compound described herein that undergoes chemicalconversion by metabolic processes before becoming an active or moreactive pharmacological agent or active compound described herein.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates, andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, ester groups (e.g., ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds, and thelike, as well as sulfides that are oxidized to form sulfoxides orsulfones.

The term “protecting group” refers to a grouping of atoms that whenattached to a reactive group in a molecule masks, reduces or preventsthat reactivity. Examples of protecting groups can be found in Green andWuts, Protective Groups in Organic Chemistry, (Wiley, 2.sup.nd ed.1991); Harrison and Harrison et al., Compendium of Synthetic OrganicMethods, Vols. 1-8 (John Wiley and Sons, 1971-1996); and Kocienski,Protecting Groups, (Verlag, 3^(rd) ed. 2003).

The term “amine protecting group” is intended to mean a functional groupthat converts an amine, amide, or other nitrogen-containing moiety intoa different chemical group that is substantially inert to the conditionsof a particular chemical reaction. Amine protecting groups arepreferably removed easily and selectively in good yield under conditionsthat do not affect other functional groups of the molecule. Examples ofamine protecting groups include, but are not limited to, formyl, acetyl,benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, t-butyloxycarbonyl(Boc), p-methoxybenzyl, methoxymethyl, tosyl, trifluoroacetyl,trimethylsilyl (TMS), fluorenyl-methyloxycarbonyl,2-trimethylsilyl-ethyoxycarbonyl, 1-methyl-1-(4-biphenylyl)ethoxycarbonyl, allyloxycarbonyl, benzyloxycarbonyl (CBZ),2-trimethylsilyl-ethanesulfonyl (SES), trityl and substituted tritylgroups, 9-fluorenylmethyloxycarbonyl (FMOC), nitro-veratryloxycarbonyl(NVOC), and the like. Those of skill in the art can identify othersuitable amine protecting groups.

Representative hydroxy protecting groups include those where the hydroxygroup is either acylated or alkylated such as benzyl, and trityl ethersas well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethersand allyl ethers.

Additionally, the salts of the compounds described herein, can exist ineither hydrated or unhydrated (the anhydrous) form or as solvates withother solvent molecules. Non-limiting examples of hydrates includemonohydrates, dihydrates, etc. Nonlimiting examples of solvates includeethanol solvates, acetone solvates, etc.

The term “solvates” means solvent addition forms that contain eitherstoichiometric or non-stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

The compounds, salts and prodrugs described herein can exist in severaltautomeric forms, including the enol and imine form, and the keto andenamine form and geometric isomers and mixtures thereof. Tautomers existas mixtures of a tautomeric set in solution. In solid form, usually onetautomer predominates. Even though one tautomer may be described, thepresent application includes all tautomers of the present compounds. Atautomer is one of two or more structural isomers that exist inequilibrium and are readily converted from one isomeric form to another.This reaction results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Insolutions where tautomerization is possible, a chemical equilibrium ofthe tautomers will be reached. The exact ratio of the tautomers dependson several factors, including temperature, solvent, and pH. The conceptof tautomers that are interconvertable by tautomerizations is calledtautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs.

Tautomerizations can be catalyzed by: Base: 1. deprotonation; 2.formation of a delocalized anion (e.g., an enolate); 3. protonation at adifferent position of the anion; Acid: 1. protonation; 2. formation of adelocalized cation; 3. deprotonation at a different position adjacent tothe cation.

The term “analogue” refers to a chemical compound that is structurallysimilar to another but differs slightly in composition (as in thereplacement of one atom by an atom of a different element or in thepresence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analogue is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

A “patient,” “subject,” or “host” to be treated by the subject methodmay mean either a human or non-human animal, such as a mammal, a fish, abird, a reptile, or an amphibian. Thus, the subject of the hereindisclosed methods can be a human, non-human primate, horse, pig, rabbit,dog, sheep, goat, cow, cat, guinea pig or rodent. The term does notdenote a particular age or sex. Thus, adult and newborn subjects, aswell as fetuses, whether male or female, are intended to be covered. Inone aspect, the subject is a mammal. A patient refers to a subjectafflicted with a disease or disorder.

The terms “prophylactic” or “therapeutic” treatment is art-recognizedand includes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The terms “therapeutic agent”, “drug”, “medicament” and “bioactivesubstance” are art-recognized and include molecules and other agentsthat are biologically, physiologically, or pharmacologically activesubstances that act locally or systemically in a patient or subject totreat a disease or condition. The terms include without limitationpharmaceutically acceptable salts thereof and prodrugs. Such agents maybe acidic, basic, or salts; they may be neutral molecules, polarmolecules, or molecular complexes capable of hydrogen bonding; they maybe prodrugs in the form of ethers, esters, amides and the like that arebiologically activated when administered into a patient or subject.

The phrase “therapeutically effective amount” or “pharmaceuticallyeffective amount” is an art-recognized term. In certain embodiments, theterm refers to an amount of a therapeutic agent that produces somedesired effect at a reasonable benefit/risk ratio applicable to anymedical treatment. In certain embodiments, the term refers to thatamount necessary or sufficient to eliminate, reduce or maintain a targetof a particular therapeutic regimen. The effective amount may varydepending on such factors as the disease or condition being treated, theparticular targeted constructs being administered, the size of thesubject or the severity of the disease or condition. One of ordinaryskill in the art may empirically determine the effective amount of aparticular compound without necessitating undue experimentation. Incertain embodiments, a therapeutically effective amount of a therapeuticagent for in vivo use will likely depend on a number of factors,including: the rate of release of an agent from a polymer matrix, whichwill depend in part on the chemical and physical characteristics of thepolymer; the identity of the agent; the mode and method ofadministration; and any other materials incorporated in the polymermatrix in addition to the agent.

The term “ED50” is art-recognized. In certain embodiments, ED50 meansthe dose of a drug, which produces 50% of its maximum response oreffect, or alternatively, the dose, which produces a pre-determinedresponse in 50% of test subjects or preparations. The term “LD50” isart-recognized. In certain embodiments, LD50 means the dose of a drug,which is lethal in 50% of test subjects. The term “therapeutic index” isan art-recognized term, which refers to the therapeutic index of a drug,defined as LD50/ED50.

The terms “IC₅₀,” or “half maximal inhibitory concentration” is intendedto refer to the concentration of a substance (e.g., a compound or adrug) that is required for 50% inhibition of a biological process, orcomponent of a process, including a protein, subunit, organelle,ribonucleoprotein, etc.

With respect to any chemical compounds, the present application isintended to include all isotopes of atoms occurring in the presentcompounds. Isotopes include those atoms having the same atomic numberbut different mass numbers. By way of general example and withoutlimitation, isotopes of hydrogen include tritium and deuterium, andisotopes of carbon include C-13 and C-14.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent can be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent can be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

When an atom or a chemical moiety is followed by a subscripted numericrange (e.g., C₁₋₆), it is meant to encompass each number within therange as well as all intermediate ranges. For example, “C₁₋₆ alkyl” ismeant to include alkyl groups with 1, 2, 3, 4, 5, 6, 1-6, 1-5, 1-4, 1-3,1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, and 5-6 carbons.

The term “alkyl” is intended to include both branched (e.g., isopropyl,tert-butyl, isobutyl), straight-chain e.g., methyl, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl), and cycloalkyl(e.g., alicyclic) groups (e.g., cyclopropyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. Such aliphatic hydrocarbon groupshave a specified number of carbon atoms. For example, C₁₋₆ alkyl isintended to include C₁, C₂, C₃, C₄, C₅, and C₆ alkyl groups. As usedherein, “lower alkyl” refers to alkyl groups having from 1 to 6 carbonatoms in the backbone of the carbon chain. “Alkyl” further includesalkyl groups that have oxygen, nitrogen, sulfur or phosphorous atomsreplacing one or more hydrocarbon backbone carbon atoms. In certainembodiments, a straight chain or branched chain alkyl has six or fewercarbon atoms in its backbone (e.g., C₁-C₆ for straight chain, C₃-C₆ forbranched chain), for example four or fewer. Likewise, certaincycloalkyls have from three to eight carbon atoms in their ringstructure, such as five or six carbons in the ring structure.

The term “substituted alkyls” refers to alkyl moieties havingsubstituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone. Such substituents can include, for example, alkyl,alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Cycloalkyls can be further substituted, e.g.,with the substituents described above. An “alkylaryl” or an “aralkyl”moiety is an alkyl substituted with an aryl (e.g., phenylmethyl(benzyl)). If not otherwise indicated, the terms “alkyl” and “loweralkyl” include linear, branched, cyclic, unsubstituted, substituted,and/or heteroatom-containing alkyl or lower alkyl, respectively.

The term “alkenyl” refers to a linear, branched or cyclic hydrocarbongroup of 2 to about 24 carbon atoms containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl,cyclopentenyl, cyclohexenyl, cyclooctenyl, and the like. Generally,although again not necessarily, alkenyl groups can contain 2 to about 18carbon atoms, and more particularly 2 to 12 carbon atoms. The term“lower alkenyl” refers to an alkenyl group of 2 to 6 carbon atoms, andthe specific term “cycloalkenyl” intends a cyclic alkenyl group,preferably having 5 to 8 carbon atoms. The term “substituted alkenyl”refers to alkenyl substituted with one or more substituent groups, andthe terms “heteroatom-containing alkenyl” and “heteroalkenyl” refer toalkenyl or heterocycloalkenyl (e.g., heterocylcohexenyl) in which atleast one carbon atom is replaced with a heteroatom. If not otherwiseindicated, the terms “alkenyl” and “lower alkenyl” include linear,branched, cyclic, unsubstituted, substituted, and/orheteroatom-containing alkenyl and lower alkenyl, respectively.

The term “alkynyl” refers to a linear or branched hydrocarbon group of 2to 24 carbon atoms containing at least one triple bond, such as ethynyl,n-propynyl, and the like. Generally, although again not necessarily,alkynyl groups can contain 2 to about 18 carbon atoms, and moreparticularly can contain 2 to 12 carbon atoms. The term “lower alkynyl”intends an alkynyl group of 2 to 6 carbon atoms. The term “substitutedalkynyl” refers to alkynyl substituted with one or more substituentgroups, and the terms “heteroatom-containing alkynyl” and“heteroalkynyl” refer to alkynyl in which at least one carbon atom isreplaced with a heteroatom. If not otherwise indicated, the terms“alkynyl” and “lower alkynyl” include linear, branched, unsubstituted,substituted, and/or heteroatom-containing alkynyl and lower alkynyl,respectively.

The terms “alkyl”, “alkenyl”, and “alkynyl” are intended to includemoieties which are diradicals, i.e., having two points of attachment. Anonlimiting example of such an alkyl moiety that is a diradical is—CH₂CH₂—, i.e., a C₂ alkyl group that is covalently bonded via eachterminal carbon atom to the remainder of the molecule.

The term “alkoxy” refers to an alkyl group bound through a single,terminal ether linkage; that is, an “alkoxy” group may be represented as—O-alkyl where alkyl is as defined above. A “lower alkoxy” group intendsan alkoxy group containing 1 to 6 carbon atoms, and includes, forexample, methoxy, ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc.Preferred substituents identified as “C₁-C₆ alkoxy” or “lower alkoxy”herein contain 1 to 3 carbon atoms, and particularly preferred suchsubstituents contain 1 or 2 carbon atoms (i.e., methoxy and ethoxy).

The term “aryl” refers to an aromatic substituent containing a singlearomatic ring or multiple aromatic rings that are fused together,directly linked, or indirectly linked (such that the different aromaticrings are bound to a common group such as a methylene or ethylenemoiety). Aryl groups can contain 5 to 20 carbon atoms, and particularlypreferred aryl groups can contain 5 to 14 carbon atoms. Examples of arylgroups include benzene, phenyl, pyrrole, furan, thiophene, thiazole,isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole,isooxazole, pyridine, pyrazine, pyridazine, and pyrimidine, and thelike. Furthermore, the term “aryl” includes multicyclic aryl groups,e.g., tricyclic, bicyclic, e.g., naphthalene, benzoxazole,benzodioxazole, benzothiazole, benzoimidazole, benzothiophene,methylenedioxyphenyl, quinoline, isoquinoline, napthridine, indole,benzofuran, purine, benzofuran, deazapurine, or indolizine. Those arylgroups having heteroatoms in the ring structure may also be referred toas “aryl heterocycles”, “heterocycles,” “heteroaryls” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents as described above, as forexample, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl,alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkylamino,dialkylamino, arylamino, diaryl amino, and alkylaryl amino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings, which are not aromatic so as to form amulticyclic system (e.g., tetralin, methylenedioxyphenyl). If nototherwise indicated, the term “aryl” includes unsubstituted,substituted, and/or heteroatom-containing aromatic substituents.

The term “alkaryl” refers to an aryl group with an alkyl substituent,and the term “aralkyl” refers to an alkyl group with an arylsubstituent, wherein “aryl” and “alkyl” are as defined above. Exemplaryaralkyl groups contain 6 to 24 carbon atoms, and particularly preferredaralkyl groups contain 6 to 16 carbon atoms. Examples of aralkyl groupsinclude, without limitation, benzyl, 2-phenyl-ethyl, 3-phenyl-propyl,4-phenyl-butyl, 5-phenyl-pentyl, 4-phenylcyclohexyl, 4-benzylcyclohexyl,4-phenylcyclohexylmethyl, 4-benzylcyclohexylmethyl, and the like.Alkaryl groups include, for example, p-methylphenyl, 2,4-dimethylphenyl,p-cyclohexylphenyl, 2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl,3-ethyl-cyclopenta-1,4-diene, and the like.

The terms “heterocyclyl” or “heterocyclic group” include closed ringstructures, e.g., 3- to 10-, or 4- to 7-membered rings, which includeone or more heteroatoms. “Heteroatom” includes atoms of any elementother than carbon or hydrogen. Examples of heteroatoms include nitrogen,oxygen, sulfur and phosphorus.

Heterocyclyl groups can be saturated or unsaturated and includepyrrolidine, oxolane, thiolane, piperidine, piperazine, morpholine,lactones, lactams, such as azetidinones and pyrrolidinones, sultams, andsultones. Heterocyclic groups such as pyrrole and furan can havearomatic character. They include fused ring structures, such asquinoline and isoquinoline. Other examples of heterocyclic groupsinclude pyridine and purine. The heterocyclic ring can be substituted atone or more positions with such substituents as described above, as forexample, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl,cyano, azido, heterocyclyl, or an aromatic or heteroaromatic moiety.Heterocyclic groups can also be substituted at one or more constituentatoms with, for example, a lower alkyl, a lower alkenyl, a lower alkoxy,a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, ahydroxyl, —CF₃, or —CN, or the like.

The term “halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.“Counterion” is used to represent a small, negatively charged speciessuch as fluoride, chloride, bromide, iodide, hydroxide, acetate, andsulfate. The term sulfoxide refers to a sulfur attached to 2 differentcarbon atoms and one oxygen and the S—O bond can be graphicallyrepresented with a double bond (S═O), a single bond without charges(S—O) or a single bond with charges [S(+)—O(−)].

The terms “substituted” as in “substituted alkyl,” “substituted aryl,”and the like, as alluded to in some of the aforementioned definitions,is meant that in the alkyl, aryl, or other moiety, at least one hydrogenatom bound to a carbon (or other) atom is replaced with one or morenon-hydrogen substituents. Examples of such substituents include,without limitation: functional groups such as halo, hydroxyl, silyl,sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄ alkoxycarbonyl(—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀ arylcarbonato (—O—(CO)—O-aryl),carboxy (—COOH), carboxylato (—COO—), carbamoyl (—(CO)—NH₂),mono-(C₁-C₂₄ alkyl)-substituted carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)),di-(C₁-C₄ alkyl)-substituted carbamoyl (—(CO)—N(C₁-C₂₄ alkyl)₂),mono-substituted arylcarbamoyl (—(CO)—NH-aryl), thiocarbamoyl(—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano (—CN), isocyano (—N⁺C⁻),cyanato (—O—CN), isocyanato (—ON⁺C⁻), isothiocyanato (—S—CN), azido(—N═N⁺═N⁻), formyl (—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), mono-and di-(C₁-C₂₄ alkyl)-substituted amino, mono- and di-(C₅-C₂₀aryl)-substituted amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀arylamido (—NH—(CO)-aryl), imino (—CR═NH where R=hydrogen, C₁-C₂₄ alkyl,C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), alkylimino(—CR═N(alkyl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), arylimino(—CR═N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro(—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl(—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl (—(SO)-alkyl),C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄ alkylsulfonyl (—SO₂-alkyl),C₅-C₂₀ arylsulfonyl (—SO₂-aryl), phosphono (—P(O)(OH)₂), phosphonato(—P(O)(O)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), and phosphino(—PH₂); and the hydrocarbyl moieties C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, and C₆-C₂₄ aralkyl.

In addition, the aforementioned functional groups may, if a particulargroup permits, be further substituted with one or more additionalfunctional groups or with one or more hydrocarbyl moieties such as thosespecifically enumerated above. Analogously, the above-mentionedhydrocarbyl moieties may be further substituted with one or morefunctional groups or additional hydrocarbyl moieties such as thosespecifically enumerated.

When the term “substituted” appears prior to a list of possiblesubstituted groups, it is intended that the term apply to every memberof that group. For example, the phrase “substituted alkyl, alkenyl, andaryl” is to be interpreted as “substituted alkyl, substituted alkenyl,and substituted aryl.” Analogously, when the term“heteroatom-containing” appears prior to a list of possibleheteroatom-containing groups, it is intended that the term apply toevery member of that group. For example, the phrase“heteroatom-containing alkyl, alkenyl, and aryl” is to be interpreted as“heteroatom-containing alkyl, substituted alkenyl, and substituted aryl.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anon-hydrogen substituent may or may not be present on a given atom, and,thus, the description includes structures wherein a non-hydrogensubstituent is present and structures wherein a non-hydrogen substituentis not present.

The terms “stable compound” and “stable structure” are meant to indicatea compound that is sufficiently robust to survive isolation, and asappropriate, purification from a reaction mixture, and formulation intoan efficacious therapeutic agent.

The terms “free compound” is used herein to describe a compound in theunbound state.

Throughout the description, where compositions are described as having,including, or comprising, specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the compositionsand methods described herein remains operable. Moreover, two or moresteps or actions can be conducted simultaneously.

The term “small molecule” is an art-recognized term. In certainembodiments, this term refers to a molecule, which has a molecularweight of less than about 2000 amu, or less than about 1000 amu, andeven less than about 500 amu.

All percentages and ratios used herein, unless otherwise indicated, areby weight.

The term “neoplasm” refers to any abnormal mass of cells or tissue as aresult of neoplasia. The neoplasm may be benign, potentially malignant(precancerous), or malignant (cancerous). An adenoma is an example of aneoplasm.

As used herein, the phrase “therapeutically- orpharmaceutically-effective amount” as applied to the disclosedcompositions refers to the amount of composition sufficient to induce adesired biological result. That result can be alleviation of the signs,symptoms, or causes of a disease, or any other desired alteration of abiological system. For example, the result can involve a decrease and/orreversal of cancerous cell growth.

As used herein, the terms “inhibit”, “inhibiting”, or “inhibition”includes any measurable reproducible substantial reduction in theinteraction between Bcl-2 and IP₃R, cancer, or any other activitiesBcl-2 may mediate. A substantial reduction is a “reproducible”, i.e.,consistently observed, reduction in binding.

Embodiments described herein relate to therapeutic compounds,pharmaceutical compositions comprising the compounds, the use of thecompounds in methods of inhibiting Bcl-2 binding to inositol1,4,5-triphosphate receptors (IP₃R), and to the use of the compoundsderived from the Bcl-2 binding domain of IP₃R in methods of inducingapoptosis in cells expressing Bcl-2 and IP₃R, particularly to inducingapoptosis in neoplastic cells (e.g., cancer cells, such as chroniclymphocytic leukemia or multiple myeloma) expressing Bcl-2 and IP₃R.

Bcl-2 interacts directly with the activation coupling domain of IP₃R.The coupling domain is necessary to keep the IP₃R channel closed andregulates the activity of IP₃R by binding to regulatory proteins. Bybinding to this region, Bcl-2 exerts its regulatory effect onIP₃-mediated Ca²⁺ signals. Compounds derived from the specificBcl-2-interacting domain of IP₃R, such as a BH4 domain binding peptide,can mimic IP₃R's binding effect and when administered to a neoplasticcell expressing Bcl-2 and IP₃R induce apoptosis and/or necrosis in theneoplastic cell. For example, the Bcl-2-IP₃R interaction inhibitorBIRD-2 (Bcl-2-IP3R interaction Disrupter-2) is a synthetic BH4 domainbinding peptide derived from the Bcl-2 binding site on the IP₃R (SeeFIG. 2).

Compounds or therapeutic agents described herein, which are capable ofbinding to the BH4 domain of Bcl-2 and mimicking the Bcl-2-IP₃Rinteraction inhibitory action of BIRD-2 (i.e., a BIRD-2 mimetic agent)to induce apoptosis in neoplastic cell expressing Bcl-2 and IP₃R, suchas multiple myeloma cells and primary human Chronic lymphocytic leukemia(CLL) cells, can be identified using high throughput screening and invitro assays, described in the Example below.

The compounds or therapeutic agents described herein can be any smallchemical molecule or compound that can inhibit binding of Bcl-2 andIP₃R. Typically, test compounds will be small chemical molecules,natural products, or peptides. The assays are designed to screen largechemical libraries by automating the assay steps and providing compoundsfrom any convenient source to assays, which are typically run inparallel (e.g., in microtiter formats on microtiter plates in roboticassays).

In some embodiments, the compound can be derived from the Bcl-2 bindingdomain of IP₃R. The Bcl-2 binding domain of IP₃R can include, forexample, the BH4 binding domain of IP₃R. In some aspects, the compoundcan include a BIRD-2 mimetic agent. In other aspects, the compound caninclude a non-peptidic low molecular weight BIRD-2 mimetic agent.

In some embodiments, the compound or inhibitor of Bcl-2 binding to IP₃Rcan be a compound with the following general formula (I):

-   -   wherein X¹ is CH₂, or C═O;    -   R¹, R², R³, R⁴, and R⁵ are the same or different and are        independently selected from the group consisting of hydrogen,        substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,        C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heterocycloalkenyl containing from        5-7 ring atoms, (wherein from 1-3 of the ring atoms is        independently selected from N, NH, N(C₁-C₆ alkyl), NC(O) (C₁-C₆        alkyl), O, and S), heteroaryl or heterocyclyl containing from        5-14 ring atoms, (wherein from 1-6 of the ring atoms is        independently selected from N, NH, N(C₁-C₃ alkyl), O, and S),        C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl,        sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy,        C₅-C₂₀ aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl)        and C₆-C₂₀ arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄        alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl        (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀        arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato        (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl        (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),        thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano        (—CN), isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻),        isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H),        thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀        aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido        (—NH—(CO)-aryl), sulfanamido (—SO₂N(R)₂ where R is independently        H, alkyl, aryl or heteroaryl), imino (—CR═NH where R is        hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen,        alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl),        where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂),        nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄        alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl        (—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl        (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄        alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),        sulfonamide (—SO₂—NH₂, —SO₂NY₂ (wherein Y is independently H,        arlyl or alkyl), phosphono (—P(O)(OH)₂), phosphonato        (—P(O)(O)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), phosphino        (—PH₂), polyalkyl ethers (—[(CH₂)_(n)O]_(m)), phosphates,        phosphate esters [—OP(O)(OR)₂ where R═H, methyl or other alkyl],        groups incorporating amino acids or other moieties expected to        bear positive or negative charge at physiological pH, and        combinations thereof; and pharmaceutically acceptable salts        thereof.

In other embodiments, the inhibitor of Bcl-2 binding to IP₃R can be acompound with the following general formula (II):

-   -   wherein X¹ is CH₂, or C═O;    -   R¹, R², R³, and R⁴ are the same or different and are        independently selected from the group consisting of hydrogen,        substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,        C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heterocycloalkenyl containing from        5-7 ring atoms, (wherein from 1-3 of the ring atoms is        independently selected from N, NH, N(C₁-C₆ alkyl), NC(O) (C₁-C₆        alkyl), O, and S), heteroaryl or heterocyclyl containing from        5-14 ring atoms, (wherein from 1-6 of the ring atoms is        independently selected from N, NH, N(C₁-C₃ alkyl), O, and S),        C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl,        sulfhydryl, C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy,        C₅-C₂₀ aryloxy, acyl (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl)        and C₆-C₂₀ arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄        alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl        (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀        arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato        (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl        (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),        thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano        (—CN), isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻),        isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H),        thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀        aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido        (—NH—(CO)-aryl), sulfanamido (—SO₂N(R)₂ where R is independently        H, alkyl, aryl or heteroaryl), imino (—CR═NH where R is        hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen,        alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl),        where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂),        nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄        alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl        (—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl        (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄        alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),        sulfonamide (—SO₂—NH₂, —SO₂NY₂ (wherein Y is independently H,        arlyl or alkyl), phosphono (—P(O)(OH)₂), phosphonato        (—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), phosphino        (—PH₂), polyalkyl ethers (—[(CH₂)_(n)O]_(m)), phosphates,        phosphate esters [—OP(O)(OR)₂ where R═H, methyl or other alkyl],        groups incorporating amino acids or other moieties expected to        bear positive or negative charge at physiological pH, and        combinations thereof;    -   R⁶ is a halo (e.g., Br, Cl, or F), n¹ is 0-3, and        pharmaceutically acceptable salts thereof.

In other embodiments, the inhibitor of Bcl-2 binding to IP₃R can be acompound with the following general formula (III):

-   -   wherein X¹ is CH₂, or C═O;    -   R¹, R², and R⁴ are the same or different and are independently        selected from the group consisting of hydrogen, substituted or        unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,        C₃-C₂₀ aryl, heterocycloalkenyl containing from 5-7 ring atoms,        (wherein from 1-3 of the ring atoms is independently selected        from N, NH, N(C₁-C₆ alkyl), NC(O) (C₁-C₆ alkyl), O, and S),        heteroaryl or heterocyclyl containing from 5-14 ring atoms,        (wherein from 1-6 of the ring atoms is independently selected        from N, NH, N(C₁-C₃ alkyl), O, and S), C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy,        C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl        (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀        arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄        alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl        (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀        arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato        (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl        (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),        thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano        (—CN), isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻),        isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H),        thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀        aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido        (—NH—(CO)-aryl), sulfanamido (—SO₂N(R)₂ where R is independently        H, alkyl, aryl or heteroaryl), imino (—CR═NH where R is        hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen,        alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl),        where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂),        nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄        alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl        (—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl        (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄        alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),        sulfonamide (—SO₂—NH₂, —SO₂NY₂ (wherein Y is independently H,        arlyl or alkyl), phosphono (—P(O)(OH)₂), phosphonato        (—P(O)(O)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), phosphino        (—PH₂), polyalkyl ethers (—[(CH₂)_(n)O]_(m)), phosphates,        phosphate esters [—OP(O)(OR)₂ where R═H, methyl or other alkyl],        groups incorporating amino acids or other moieties expected to        bear positive or negative charge at physiological pH, and        combinations thereof;    -   R⁶ is a halo (e.g., Br, Cl, or F), n¹ is 0-3, and        pharmaceutically acceptable salts thereof.

In other embodiments, the inhibitor of Bcl-2 binding to IP₃R can be acompound with the following general formula (IV):

-   -   wherein X¹ is CH₂, or C═O;    -   R¹, R³, and R⁴ are the same or different and are independently        selected from the group consisting of hydrogen, substituted or        unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl, C₂-C₂₄ alkynyl,        C₃-C₂₀ aryl, heterocycloalkenyl containing from 5-7 ring atoms,        (wherein from 1-3 of the ring atoms is independently selected        from N, NH, N(C₁-C₆ alkyl), NC(O) (C₁-C₆ alkyl), O, and S),        heteroaryl or heterocyclyl containing from 5-14 ring atoms,        (wherein from 1-6 of the ring atoms is independently selected        from N, NH, N(C₁-C₃ alkyl), O, and S), C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, halo, silyl, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy,        C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy, acyl        (including C₂-C₂₄ alkylcarbonyl (—CO-alkyl) and C₆-C₂₀        arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl), C₂-C₂₄        alkoxycarbonyl (—(CO)—O-alkyl), C₆-C₂₀ aryloxycarbonyl        (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato (—O—(CO)—O-alkyl), C₆-C₂₀        arylcarbonato (—O—(CO)—O-aryl), carboxy (—COOH), carboxylato        (—COO⁻), carbamoyl (—(CO)—NH₂), C₁-C₂₄ alkyl-carbamoyl        (—(CO)—NH(C₁-C₂₄ alkyl)), arylcarbamoyl (—(CO)—NH-aryl),        thiocarbamoyl (—(CS)—NH₂), carbamido (—NH—(CO)—NH₂), cyano        (—CN), isocyano (—N⁺C⁻), cyanato (—O—CN), isocyanato (—O—N⁺═C⁻),        isothiocyanato (—S—CN), azido (—N═N⁺═N⁻), formyl (—(CO)—H),        thioformyl (—(CS)—H), amino (—NH₂), C₁-C₂₄ alkyl amino, C₅-C₂₀        aryl amino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₀ arylamido        (—NH—(CO)-aryl), sulfanamido (—SO₂N(R)₂ where R is independently        H, alkyl, aryl or heteroaryl), imino (—CR═NH where R is        hydrogen, C₁-C₂₄ alkyl, C₅-C₂₀ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄        aralkyl, etc.), alkylimino (—CR═N(alkyl), where R=hydrogen,        alkyl, aryl, alkaryl, aralkyl, etc.), arylimino (—CR═N(aryl),        where R=hydrogen, alkyl, aryl, alkaryl, etc.), nitro (—NO₂),        nitroso (—NO), sulfo (—SO₂—OH), sulfonato (—SO₂—O⁻), C₁-C₂₄        alkylsulfanyl (—S-alkyl; also termed “alkylthio”), arylsulfanyl        (—S-aryl; also termed “arylthio”), C₁-C₂₄ alkylsulfinyl        (—(SO)-alkyl), C₅-C₂₀ arylsulfinyl (—(SO)-aryl), C₁-C₂₄        alkylsulfonyl (—SO₂-alkyl), C₅-C₂₀ arylsulfonyl (—SO₂-aryl),        sulfonamide (—SO₂—NH₂, —SO₂NY₂ (wherein Y is independently H,        arlyl or alkyl), phosphono (—P(O)(OH)₂), phosphonato        (—P(O)(O⁻)₂), phosphinato (—P(O)(O⁻)), phospho (—PO₂), phosphino        (—PH₂), polyalkyl ethers (—[(CH₂)_(n)O]_(m)), phosphates,        phosphate esters [—OP(O)(OR)₂ where R═H, methyl or other alkyl],        groups incorporating amino acids or other moieties expected to        bear positive or negative charge at physiological pH, and        combinations thereof;    -   R⁶ is a halo (e.g., Br, Cl, or F), n¹ is 0-3, and        pharmaceutically acceptable salts thereof.

In some embodiments, the inhibitor of Bcl-2 binding to IP₃R can includea compound having the formula selected from the group consisting of:

and pharmaceutically acceptable salts thereof.

In another aspect, the therapeutic agent can include compounds havingthe following formulas:

and pharmaceutically acceptable salts thereof.

In other embodiments, the inhibitor of Bcl-2 binding to IP₃R can includea compound having the formula:

and pharmaceutically acceptable salts thereof.

In still other embodiments, the inhibitor of Bcl-2 binding to IP₃R caninclude a compound having the formula:

and pharmaceutically acceptable salts thereof.

The therapeutic agents described above can be used for treatment of adisease or condition in a human where inhibition of Bcl-2-IP₃Rinteraction and thereby the induction of apoptosis, such as in humanneoplastic cells that express IP₃R and Bcl-2 is beneficial. In someembodiments, the therapeutic agents can be used in a method of treatinga neoplastic disorder in a subject. The method includes administering toneoplastic cells of the subject expressing IP₃R and Bcl-2 atherapeutically effective amount of a Bcl-2-IP₃R interaction inhibitorycompound that inhibits binding of Bcl-2 to IP₃ receptors (IP₃R) of cellsthat express IP₃R and Bcl-2. The compound or therapeutic agent can beadministered in association with one or more non-toxic, pharmaceuticallyacceptable carriers and/or diluents and/or adjuvants and if desiredother active ingredients.

The compounds or therapeutic agents described herein can be used totreat conditions and diseases including but not limited to Bcl-2associated cancers. Bcl-2 associated cancer can include cancerassociated with malignant transformation of B-lymphocytes, small celllung cancer, and pancreatic cancer. In some embodiments, Bcl-2associated cancers associated with malignant transformation ofB-lymphocytes include but are not limited to chronic lymphocyticleukemia (CLL), follicular lymphoma, diffuse large B-cell lymphoma, andmultiple myeloma (MM).

In some embodiments, the therapeutic agents described herein can be usedfor the treatment of a Bcl-2 associated cancer that is resistant to ananti-cancer agent, such as multiple myeloma. In certain embodiments, thetherapeutic agents described above can be used for the treatment of aBcl-2 associated cancer that is resistant to the ABT class ofBH-3-mimetic agents.

The therapeutic agents described herein can be provided in the form ofpharmaceutical compositions. The pharmaceutical compositions can beadministered to any animal that can experience the beneficial effects ofthe compounds of the invention. Foremost among such animals are humans,although the invention is not intended to be so limited.

The pharmaceutical compositions will generally comprise an effectiveamount of the Bcl-2-IP₃R interaction inhibitory compound, dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.Combined therapeutics are also contemplated, and the same type ofunderlying pharmaceutical compositions may be employed for both singleand combined medicaments.

In addition to the pharmacologically active compounds, thepharmaceutical preparations of the compounds can contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries that facilitate processing of the active compounds intopreparations that can be used pharmaceutically. The pharmaceuticalpreparations can be manufactured in a manner that is, itself, known, forexample, by means of conventional mixing, granulating, dragee-making,dissolving, or lyophilizing processes. Thus, pharmaceutical preparationsfor oral use can be obtained by combining the active compounds withsolid excipients, optionally grinding the resulting mixture andprocessing the mixture of granules, after adding suitable auxiliaries,if desired or necessary, to obtain tablets or dragee cores.

The Bcl-2-IP₃R interaction inhibitory compounds will most often beformulated for parenteral administration, e.g., formulated for injectionvia the intravenous, intramuscular, sub-cutaneous, transdermal, or othersuch routes, including peristaltic administration and directinstillation into a tumor or disease site (intracavity administration).The preparation of an aqueous composition that contains such a compoundas an active ingredient will be known to those of skill in the art inlight of the present disclosure. Typically, such compositions can beprepared as injectables, either as liquid solutions or suspensions;solid forms suitable for using to prepare solutions or suspensions uponthe addition of a liquid prior to injection can also be prepared; andthe preparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form should be sterile and fluid to theextent that syringability exists. It should be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms, such as bacteria and fungi.

Compositions of the Bcl-2-IP₃R interaction inhibitory compounds can beformulated into a sterile aqueous composition in a neutral or salt form.Solutions as free base or pharmacologically acceptable salts can beprepared in water suitably mixed with a surfactant, such ashydroxypropylcellulose. Pharmaceutically acceptable salts, include theacid addition salts (formed with the free amino groups of the protein),and those that are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,trifluoroacetic, oxalic, tartaric, mandelic, and the like. Salts formedwith the free carboxyl groups can also be derived from inorganic basessuch as, for example, sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,histidine, procaine and the like.

Carriers can include solvents and dispersion media containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride. Theproper fluidity can be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants.

Under ordinary conditions of storage and use, all such preparationsshould contain a preservative to prevent the growth of microorganisms.The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Prior to or upon formulation, the Bcl-2-IP₃R interaction inhibitorycompounds can be extensively dialyzed to remove undesired smallmolecular weight molecules, and/or lyophilized for more readyformulation into a desired vehicle, where appropriate. Sterileinjectable solutions are prepared by incorporating the active agents inthe required amount in the appropriate solvent with various of the otheringredients enumerated above, as desired, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle thatcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above.

Pharmaceutical compositions can generally include an amount of aBcl-2-IP₃R interaction inhibitory compound admixed with an acceptablepharmaceutical diluent or excipient, such as a sterile aqueous solution,to give a range of final concentrations, depending on the intended use.The techniques of preparation are generally well known in the art asexemplified by Remington's Pharmaceutical Sciences, 16th Ed. MackPublishing Company, 1980, incorporated herein by reference. Moreover,for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards. Upon formulation, the polypeptide orconjugate solutions will be administered in a manner compatible with thedosage formulation and in such amount as is therapeutically effective.

Formulations of Bcl-2-IP₃R interaction inhibitory compounds are easilyadministered in a variety of dosage forms, such as the type ofinjectable solutions described above, but other pharmaceuticallyacceptable forms are also contemplated, e.g., tablets, pills, capsulesor other solids for oral administration, suppositories, pessaries, nasalsolutions or sprays, aerosols, inhalants, topical formulations,liposomal forms and the like. The type of form for administration willbe matched to the disease or disorder to be treated.

Pharmaceutical “slow release” capsules or “sustained release”compositions or preparations may be used and are generally applicable.Slow release formulations are generally designed to give a constant druglevel over an extended period and may be used to deliver the Bcl-2-IP₃Rinteraction inhibitory compounds in accordance with the presentinvention. The slow release formulations are typically implanted in thevicinity of the disease site, for example, at the site of a tumor.

Examples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the polypeptide orimmunoconjugate, which matrices are in the form of shaped articles,e.g., films or microcapsule. Examples of sustained-release matricesinclude polyesters; hydrogels, for example,poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol); polylactides,e.g., U.S. Pat. No. 3,773,919; copolymers of L-glutamic acid and yethyl-L-glutamate; non-degradable ethylene-vinyl acetate; degradablelactic acid-glycolic acid copolymers, such as the LUPRON DEPOT(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate); and poly-D-(−)-3-hydroxybutyric acid.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulatedpolypeptides remain in the body for a long time, they may denature oraggregate as a result of exposure to moisture at 37° C., thus reducingbiological activity and/or changing immunogenicity. Rational strategiesare available for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism involves intermolecular S—S bondformation through thio-disulfide interchange, stabilization is achievedby modifying sulfhydryl residues, lyophilizing from acidic solutions,controlling moisture content, using appropriate additives, developingspecific polymer matrix compositions, and the like.

In certain embodiments, liposomes and/or nanoparticles may also beemployed with the Bcl-2-IP₃R interaction inhibitory compounds. Theformation and use of liposomes is generally known to those of skill inthe art, as summarized below.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

Phospholipids can form a variety of structures other than liposomes whendispersed in water, depending on the molar ratio of lipid to water. Atlow ratios, the liposome is the preferred structure. The physicalcharacteristics of liposomes depend on pH, ionic strength and thepresence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) should be designed usingpolymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles may beare easily made.

A population of cells or tissues that express IP₃R and Bcl-2 can thus becontacted with a biologically or therapeutically effective amount ofBcl-2-IP₃R interaction inhibitory compound in a pharmaceutical carrierunder conditions effective to substantially inhibit Bcl-2 binding toIP₃R.

In some embodiments, the Bcl-2-IP₃R interaction inhibitory compoundsdescribed herein may be used to treat animals, patients, or subjectswith a number of neoplastic diseases, including but not limited tolymphoma (e.g., follicular B-cell lymphoma), leukemia (chroniclymphocytic leukemia), multiple myeloma, melanoma, breast, prostate, andlung carcinomas. The Bcl-2-IP₃R interaction inhibitory compounds canalso be used for reducing resistance to conventional cancer treatment.

In designing appropriate doses of the Bcl-2-IP₃R interaction inhibitorycompounds for the treatment of vascularized tumors, one may readilyextrapolate from the knowledge in the literature in order to arrive atappropriate doses for clinical administration. To achieve a conversionfrom animal to human doses, one would account for the mass of the agentsadministered per unit mass of the experimental animal and, preferably,account for the differences in the body surface area (m²) between theexperimental animal and the human patient. All such calculations arewell known and routine to those of ordinary skill in the art.

The intention of the therapeutic regimens described herein is generallyto produce significant anti-neoplastic effects while still keeping thedose below the levels associated with unacceptable toxicity. In additionto varying the dose itself, the administration regimen can also beadapted to optimize the treatment strategy. In administering theparticular doses, one can provide a pharmaceutically acceptablecomposition (according to FDA standards of sterility, pyrogenicity,purity and general safety) to the patient systemically. Intravenousinjection is generally preferred. Continuous infusion over a time periodof about 1 or 2 hours or so is also contemplated. In certainembodiments, the agent can be delivered to cancer cells by site-specificmeans.

The Bcl-2-IP₃R interaction inhibitory compounds may also be delivered incombination with a second agent that induces apoptosis in neoplasticcells. Although many anti-cancer agents may have, as part of theirmechanism of action, an apoptosis-inducing effect, certain agents havebeen discovered, designed or selected with this as a primary mechanism,as described below.

Without being bound by theory, it is believed that the combination of aBcl-2-IP₃R interaction inhibitory compound and a Bcl-2/BH3 inhibitorovercomes Bcl-2s anti-apoptotic action by reversing two separatemechanisms by which Bcl-2 inhibits apoptosis (see FIG. 3). Therefore, insome embodiments, the second agent can be a small-molecule inhibitorthat directly binds Bcl-2/IP₃R or related anti-apoptotic proteins andinhibits the Bcl-2-BH3 domain binding to BH3 domain proteins or BH3 onlymolecules, such as BID, NOXA, PUMA, BIK, BIM, and BAD (i.e., a Bcl-2/BH3inhibitor). By targeting two different regions of Bcl-2 involved inapoptosis inhibition with the Bcl-2-IP₃R interaction inhibitorycompounds described herein, which bind to the BH4 domain of Bcl-2, andan inhibitor of Bcl-2 to BH3 domain proteins, the proapoptotic activityof the Bcl-2/BH3 inhibitors and the Bcl-2-IP₃R interaction inhibitorycompounds are enhanced. In certain embodiments, a combination of aBcl-2-IP₃R interaction inhibitory compound and a Bcl-2/BH3 inhibitorinduces synergistic cytotoxicity when administered to neoplastic cells,such as human myeloma cells (see FIG. 10).

One example of a small molecule inhibitor is gossypol or1,6,7,1′,6′,7′-Hexahydroxy-5,5′-diisopropyl-3,3′-dimethyl-[2,2′]binaphthalenyl-8,8′-dicarbaldehyde.Gossypol has the following formula:

Gossypol is found in cottonseeds originally used as an herbal medicinein China. Gossypol binds via a conserved 16 amino acid motif called aBcl-2 homology-3 (BH3) domain found on the surface of antiapoptoticBcl-2 family proteins. This binding pocket represents a regulatory site,where endogenous antagonists dock onto Bcl-2 and related antiapoptoticproteins, negating their cytoprotective activity. Proof of conceptexperiments using BH3 peptides have suggested that compounds docking atthis regulatory site on Bcl-2 and Bcl-XL effectively promote apoptosisof lymphoma and leukemia cells in vivo in mice.

Gossypol interacts with the BH3-binding pockets of 4 anti-apoptoticBcl-2 family proteins tested to date, Bcl-2, Bcl-X_(L), Bcl-B, andBfl-1, displacing BH3 peptides with an inhibitory concentration of 50%(IC₅₀) of about 0.5 μM.

Another example of small molecule inhibitor of Bcl-2 is a semisyntheticanalog of gossypol known as apogossypol or5,5′-Diisopropyl-3,3′-dimethyl-[2,2′]binaphthalenyl-1,6,7,1′,6′,7′-hexaol,which has the following general formula:

Other examples of chemical inhibitors of Bcl-2, Bcl-X_(L), and Mcl-1have been reported, most of which are currently in preclinicalevaluation, including: chromenes or chromene derivatives, such as HA14-1or2-amino-6-bromo-4-cyano-ethoxycarbonyl-methyl)-4H-chromene-3-carboxylicacid ethyl ester or other compounds disclosed in U.S. Pat. No.6,492,389; thiazolidins or thiazolidin derivatives, such as BH3I-1 or(2-[5-(4-Bromo-benzylidene)-4-oxo-2-thioxo-thiazolidin-3-yl]-3-methyl-butyricacid); benzene sulfonyl derivatives, such as BH3I-2 or(5-chloro-N-[2-chloro-4-(4-chloro-benzenesulfonyl)-phenyl]-2-hydroxy-3-iodo-benzamide);antimycin analogs, such as3-(3-Formylamino-2-hydroxy-benzoylamino)-2,6-dimethyl-4,9-dioxo-8-pentyl-[1,5]dioxonane-7-carboxylicacid isopropyl ester or Antimycin A3, and antimycin analogues disclosedin U.S. Pat. No. 7,241,804 (e.g., structures I-V); theaflavins, such as3,4,6-trihydroxy-1-(3,5,7-trihydroxy-chroman-2-yl)-benzocyclohepten-5-one;epigallechatechins (EGCGs), such as 3,4,5-Trihydroxy-benzoic acid5,7-dihydroxy-2-(3,4,5-trihydroxy-phenyl)-chroman-3-yl ester;benzenesulfonamides, such as ABT-737 orN-[4-[4-(4′-Chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl)-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide(a synthetic small-molecule inhibitor produced by NMR-guided,structure-based drug design (Abbott Laboratories, North Chicago, Ill.);indoles, such as GX15-070 (Gemin X, Montreal, Canada) or2-[5-(3,5-Dimethyl-1H-pyrrol-2-ylmethylene)-4-methoxy-5H-pyroll-2-yl]-1H-indole;dibenzodiazocines, such as 2,9-Dimethoxy-11,12-dihydro-dibenzo[c,g][1,2]diazocine 5,6-dioxide; and terphenylderivatives, such as a compound having the following formula:

Side-by-side comparisons of these chemical inhibitors of antiapoptoticBcl-2 proteins have not been reported, but their approximate rank-orderpotency with respect to affinity for the BH3 pocket of Bcl-2 orBcl-X_(L) appears to beABT-737>EGCG>theafavins>gossypol>apogossypol>HA14-1 and antimycin.Accordingly, in one example the second agent administered to the cellsis ABT-737 orN-[4-[4-(4′-Chloro-biphenyl-2-ylmethyl)-piperazin-1-yl]-benzoyl)-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide.

In some embodiments, the chemical inhibitors of antiapoptotic Bcl-2proteins are BH3 mimetics capable of competitively inhibiting BH3-onlyprotein binding to the hydrophobic grooves formed by Bcl-2 domains 1-3,inducing apoptosis in cells requiring to Bcl-2 for survival. ExemplaryBH3 mimetics include, but are not limited to, ABT-737, ABT-263, andABT-199.

The Bcl-2-IP₃R interaction inhibitory compound based treatment methodsdescribed herein may also be combined with any other methods generallyemployed in the treatment of the particular tumor, disease or disorderthat the subject exhibits. So long as a particular therapeutic approachis not known to be detrimental to the patient's condition in itself, anddoes not significantly counteract the Bcl-2-IP₃R interaction inhibitorycompound based treatment, its combination with the present invention iscontemplated.

In another aspect, a Bcl-2-IP₃R interaction inhibitory compound can beco-administered with one or more anti-cellular agents. Examplesanti-cellular agents include chemotherapeutic agents, as well ascytotoxins. Chemotherapeutic agents that can be used include: hormones,such as steroids; anti-metabolites, such as cytosine arabinoside,fluorouracil, methotrexate or aminopterin; anthracyclines; mitomycin C;vinca alkaloids; demecolcine; etoposide; mithramycin; anti-tumoralkylating agents, such as chlorambucil or melphalan. Other embodimentscan include agents such as cytokines. Basically, any anti-cellular agentmay be used.

Many forms of cancer have reports of mutations in tumor suppressorgenes, such as p53. Inactivation of p53 results in a failure to promoteapoptosis. With this failure, cancer cells progress in tumorigenesis,rather than become destined for cell death. Thus, delivery of tumorsuppressors is also contemplated for use in the present invention tostimulate cell death. Examples of tumor suppressor agents are disclosedin U.S. Pat. Nos. 5,747,469; 5,677,178; and 5,756,455; 5,750,400;5,654,155; 5,710,001; 5,756,294; 5,709,999; 5,693,473; 5,753,441;5,622,829; and 5,747,282 (each incorporated herein by reference),

Other compositions that may be administered with the Bcl-2-IP₃Rinteraction inhibitory compounds, include genes encoding the tumornecrosis factor related apoptosis inducing ligand termed TRAIL, and theTRAIL polypeptide (U.S. Pat. No. 5,763,223; incorporated herein byreference); the 24 kD apoptosis-associated protease of U.S. Pat. No.5,605,826 (incorporated herein by reference); Fas-associated factor 1,FAF1 (U.S. Pat. No. 5,750,653; incorporated herein by reference). Alsocontemplated for use in these aspects of the present invention is theprovision of interleukin-1p-converting enzyme and family members, whichare also reported to stimulate apoptosis

It will be appreciated that the therapeutic agents administered with theBcl-2-IP₃R interaction inhibitory compounds are not limited to thetherapeutic agents described above, and that other therapeutic agentsand other agents, which do not have therapeutic properties, can be used.

The following example is included to demonstrate different embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the example, which follow representtechniques discovered by the inventors to function well in the practiceof the claimed embodiments, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the claims.

Example

Novel Cancer Therapeutic Drugs Targeting the BH4 Domain of Bcl-2

Proof-of-Principle for Targeting Bcl-2-IP₃R Interaction for CancerTreatment

To selectively target Bcl-2-IP₃R interaction, we developed BIRD-2(Bcl-2-IP₃R interaction Disrupter-2), a synthetic peptide derived fromthe Bcl-2 binding site on the IP₃R and previously referred to asTAT-IDP_(DD/AA) or TAT-IDP^(S) (FIG. 2). By binding to the BH4 domain ofBcl-2, BIRD-2 disrupts Bcl-2-IP₃R interaction, reversing Bcl-2'srepressive effect on IP₃R-mediated Ca²⁺ release. Through this mechanismBIRD-2 induces high amplitude Ca²⁺ elevation, triggering apoptosis inprimary human CLL cells, and diffuse large B-cell lymphoma lines.BH3-mimetics, on the other hand, induce apoptosis by binding to thehydrophobic groove formed by Bcl-2 domains 1-3, thereby inducingapoptosis by disrupting the interaction of Bcl-2 with BH3-only proteins.BIRD-2 selectively disrupts Bcl-2-IP₃R interaction and does not disruptthe binding of pro-apoptotic proteins (e.g., Bim) by Bcl-2. Thus, BIRD-2and BH3-mimetic agents target completely different regions of the Bcl-2protein and overcome Bcl-2's anti-apoptotic action by reversing twoseparate mechanisms by which Bcl-2 inhibits apoptosis (FIG. 3). Findingthat Bcl-2 inhibits apoptosis by two distinct mechanisms involvingseparate regions of the Bcl-2 protein suggests that targeting only oneof the mechanisms may be of limited therapeutic value and argues thatefforts to target the other mechanism should be undertaken. In supportof this concept, we recently discovered that BIRD-2 induces apoptosis inABT-263/ABT-199 resistant Bcl-2 positive human myeloma cell lines(HMCLs). Moreover, we find synergistic killing of HMCLs by combiningBIRD-2 with chemotherapeutic agents used to treat patients with multiplemyeloma. Collectively, these findings advocate for developing newtherapeutic agents that function like BIRD-2 to target the Bcl-2-IP₃Rinteraction, both to circumvent resistance to BH3-mimetics andpotentially increase the therapeutic efficacy of BH3-mimetics.

Reciprocal Sensitivity of Multiple Myeloma Cells to BIRD-2 andBH3-Mimetic Agents

Multiple myeloma is a Bcl-2-positive malignancy of plasma cells derivedfrom relatively mature forms of B-lymphocytes present mainly in the bonemarrow. Multiple myeloma is nearly universally fatal and thus a majorclinical challenge in need of novel targeted therapeutic approaches.Though multiple myeloma cells lack translocation responsible forelevating Bcl-2 in follicular lymphoma, the majority of human myelomacell lines (HMCLs) have elevated Bcl-2 expression levels comparable tothose in follicular lymphoma cells. Moreover, Bcl-2 levels are reportedto increase in multiple myeloma cells following chemotherapy treatment.Therefore, treatment of multiple myeloma with BH3-mimetics is anappealing strategy. In some but not all HMCLs and primary patientsamples, the BH3-mimetic ABT-737 has demonstrated both substantialsingle-agent anti-myeloma activity and synergistic activity with variouschemotherapeutic agents. Recent studies also indicate responsiveness ofHMCLs and primary patient samples to the Bcl-2-selective BH3-mimeticABT-199.

However, multiple myeloma is a heterogeneous malignancy, both in termsof clinical course and responsiveness to treatment. The heterogeneity ofmultiple myeloma has a molecular basis and molecular abnormalities areof considerable prognostic value for patient survival. The heterogeneityof HMCLs closely reflects that of primary multiple myeloma in patientsand particular subtypes correlate with responsiveness to ABT-737 andABT-199. In general, ABT-737 sensitive lines harbor the translocationassociated with a relatively high ratio of Bcl-2 to anotheranti-apoptotic Bcl-2 family member, Mcl-1.

We found for the first time that inhibiting Bcl-2-IP₃R interaction withBIRD-2 induces apoptosis in HMCLs. Significantly, BIRD-2 inducesapoptosis in two HMCLs that are resistant to BH3-mimetic agentsincluding ABT-737, ABT-263 and ABT-199. Furthermore, one of theBIRD-2-sensitive lines, NCI-H929, corresponds to a multiple molecularsubgroup that is resistant to BH3-mimetic agents and belongs to a poorprognosis category associated with chromosomal translocation. Thus,although BH3-mimetic agents represent a major advance in cancertreatment, resistance to these agents occurs and limits their efficacy,not only in multiple myeloma but other lymphoid malignancies as well.The resistance problem is not unique to HMCLs, but is illustrated bystudies of ABT-737 and ABT-199 in primary MM cells where resistance wasdetected in more than half of primary patient samples tested. Therefore,these findings indicate the potential therapeutic importance oftargeting both of Bcl-2's anti-apoptotic mechanisms. Adding to thepotential value of targeting Bcl-2-IP₃R interaction for treatment ofmultiple myeloma is the evidence reported here of synergistic activitywhen combined with chemotherapeutic agents typically employed inmultiple myeloma treatment, and with ABT-263 and ABT-199.

Interestingly, a reciprocal relationship between sensitivity to celldeath induction by BIRD-2 and the BH3-mimetic agents is revealed in ourpresent work. We observe much higher Bim levels in cells sensitive toABT compounds and lower Bim levels in cells resistant to ABT compounds.This is consistent with the known mechanism of ABT action, which is todisplace Bim from its binding site on Bcl-2, thereby activatingmitochondria-mediated cell death. Further investigation is required toidentify mechanism(s) responsible for the increased sensitivity ofABT-resistant HMCLs to BIRD-2.

In summary, we know that BIRD-2 induces apoptosis in primary human CLLcells and in diffuse large B-cell lymphoma lines. Here we show thatBIRD-2 can similarly induce apoptosis in HMCLs, including in linesresistant to the ABT class of BH3-mimetic agents. Included among theBIRD-2-sensitive but ABT-resistant lines is NCI-H929, which isrecognized as bearing a poor prognosis molecular signature. Thesefindings emphasize the potential therapeutic value of targeting theBcl-2-IP₃R interaction to induce death of ABT-resistant cells and theneed to develop therapeutic agents that target the Bcl-2-IP₃Rinteraction.

Sensitivity of Small Cell Lung Cancer Cells to Disruption of Bcl-2-IP3RInteraction

Although most of our work has been in Bcl-2-positive lymphoidmalignancies, we have recently extended our work to show that BIRD-2induces cell death in small cell lung cancer (SCLC), a Bcl-2 positiveform of cancer that has a very high mortality rate and in which noveltreatment strategies are desperately needed. BH3-mimetics are lesseffective in SCLC than in lymphoid malignancies such as CLL. We havetreated 15 SCLC lines with BIRD-21, with greater than two-fold selectivecytotoxicity in 11 of the lines, with IC₅₀ ranging from 3-40 μM.Moreover, we find that combining the BH3-mimetic agent ABT-263 withBIRD-2 induces synergistic cytotoxicity (FIG. 4), indicating the valueof targeting both mechanisms by which Bcl-2 inhibits apoptosis.

Discovery of Four Lead Compounds that Target the Bh4 Domain of Bcl-2

The preceding findings, based on our development of a synthetic peptidethat targets Bcl-2-IP₃R interaction, provides strong proof-of-principleevidence in favor of developing a therapeutic agent that blocksBcl-2-IP₃R interaction and thus kills Bcl-2-positive cancer cells. Ahigh throughput screen was performed with the goal of identifyingdrug-like compounds that bind to the BH4 domain of Bcl-2 and mimic theaction of BIRD-2. The high throughput screening identified compoundsthat promote induction of apoptosis in two Bcl-2-positive multiplemyeloma cell lines, NCI-H929 and KMS-12-BM. These two lines span aspectrum between high and low sensitivity to BIRD-2 (i.e., BIRD-2induces apoptosis in NCI-H929 with an EC₅₀ of 5 μM and KMS-12-BM with anEC₅₀ of 25 μM). Cell death measurements employed a caspase-3/7 assay(AnaSpec) in three screening tiers (single point primary screen,triplicate hit confirmation, and triplicate dose response) withsignal-to-background consistently>10 and Z′>0.5. Activity was measuredrelative to apoptosis induction by 10 μM BIRD-2.

Of the compounds tested, 148 were identified as hits at 6.5 μM in theprimary single-point screen with activity>30% relative to apoptosisinduced by 10 μM BIRD-2 in NCI-H929 cells. These compounds, along with320 additional compounds chosen by virtual screening, were screened forhit confirmation. Also, a secondary assay for compound effect onapoptosis was performed in KMS-12-BM cells. Comparing results with thetwo different cell lines, 19 compounds induced apoptosis in NCI-H929cells at 6.5 μM, while having little to no effect on apoptosis ofKMS-12-BM cells. An additional 20 compounds demonstrated activity inboth NCI-H929 and KMS-12-BM cells, with relatively greater effect onNCI-H929 than on KMS-12-BM. Thus, a total of 39 compounds were chosenfor triplicate confirmation screening. The findings are schematicallysummarized by means of the heat map in FIG. 5. An ID number, assigned bythe chemist(s) who synthesized the compounds, is located to the left.This ID is for identification purposes only and does provide otherinformation about the compound. Columns 1 and 2 represent caspaseactivity, measured after 24 hr of treatment at UCDDC, normalized to thehighest caspase activity observed. Activity levels range from low,represented by the color purple, to high, represented by the color red.Considerable variation among different compounds and between the myelomalines is observed evident. Columns 3 and 4 in the heat map (FIG. 5)summarize results of cell death assays testing cell death induction inthe same myeloma lines in the Distelhorst laboratory using the MTSassay. In addition, the same assay was used to test the sensitivity ofprimary human CLL cells to each compound, as summarized in lane 5. Theeffect of each compound on intracellular Ca²⁺ concentration was measuredby digital imaging and results are shown as percentage of cellsresponding with a significant (more than twofold above baseline)elevation of cytoplasmic Ca²⁺ concentration. The choice of the Jurkatline for these Ca²⁺ measurements was based on our extensive evidencethat BIRD-2 binds to the BH4 domain of Bcl-2 endogenously present inthis line and that disruption of Bcl-2-IP3R interaction by BIRD-2elevates cytoplasmic Ca²⁺ in this line. An example of Ca2⁺ elevation inresponse to one of the compounds is illustrated in FIG. 6.

To further substantiate the significance of the observed elevations ofcytoplasmic Ca²⁺, we tested the effect of U73122 on the Ca²⁺ responsesto 854697 in Jurkat cells (FIG. 6). U73122 is a chemical inhibitor ofphospholipase c, the enzyme that generates IP₃. U73122 completelyblocked 854697-induced Ca²⁺ elevation, demonstrating that the Ca²⁺elevations induced by 854697 were mediated through IP₃R channel opening,and thus 854697 works ‘on-target’. Moreover, 854697 also induces Ca²⁺elevation in primary CLL cells, illustrated in FIG. 7. This resemblesthe Ca²⁺ elevation induced by BIRD-2 in our earlier studies in primaryhuman CLL cells.

Of the 39 compounds tested, 14 fulfill the criteria of both inducingcell death and Ca²⁺ elevation. The next step in compound validation wasto perform surface plasmon resonance (Biacore) to test for binding tothe BH4 domain of Bcl-2. This strategy is based on our earlier workindicating that the BH4 domain binds to IP₃Rs and, moreover, BIRD-2binds to this domain, thereby disrupting Bcl-2-IP₃R interaction. Todate, we have positive evidence of compound binding to the BH4 domain in4 compounds tested by SPR (FIG. 8). Testing of the remaining compoundsfrom the 14 lead compounds is in progress. An example of a full bindingcurve calculating Kd of 5.6 μM for compound 854697 is shown in FIG. 9.

In summary, through an iterative process we screened a library of 25,480drug-like compounds, discovering that drug-like compounds 854697,197392, 96505, 391782 bind to the BH4 domain of Bcl-2, targetingBcl-2-IP₃R interaction and inducing cell death in multiple myeloma linesand primary human CLL cells.

Synergy Between 854697 and the BH3-Mimetic ABT-263

Since Bcl-2 works by two distinct mechanisms to inhibit apoptosis, weinvestigated the possibility that 854697 and ABT-263, which selectivelytarget each of these mechanisms, might synergistically kill cancercells. As shown in FIG. 10, we incubated KMS-12-BM human myeloma cellswith each compound at a fixed concentration ratio and assessed cellviability by CellTiter-Glo assay. Results indicate an increase in celldeath responses when both compounds are combined. Combination indices(CI) were computed at each concentration to determine whether theincreased cytotoxicity is due to an additive (CI=1) or synergistic(CI<1) effect. At several compound concentrations, the CI was <1,indicating synergistic cell death induction when these agents that actby different mechanisms are combined. These findings indicate additionalvalue in developing 854697 as a novel therapeutic agent forBcl-2-positive malignancies.

Finally, we have data indicating that 854697 kills small cell lungcancer cells. Importantly, 854697 does not have toxic activity towardnormal cells, as represented by normal human peripheral bloodlymphocytes.

While this invention has been shown and described with references tovarious embodiments thereof, it will be understood by those skilled inthe art that changes in form and details may be made therein withoutdeparting from the scope of the invention encompassed by the appendedclaims. All patents, publications and references cited in the foregoingspecification are herein incorporated by reference in their entirety.

Having described the invention, I claim the following:
 1. A method ofinducing apoptosis in a cell expressing Bcl-2 and IP₃R, comprising:administering to the cell a therapeutically effective amount of acompound that inhibits binding of Bcl-2 to IP₃ receptors (IP₃R) of cellsthat express IP₃R and Bcl-2, wherein the compound is derived from theBcl-2 binding domain of IP₃R; wherein the compound has the generalformula (I):

wherein X¹ is CH₂, or C═O; R¹, R², R³, R⁴, and R⁵ are the same ordifferent and are independently selected from the group consisting ofhydrogen, substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heterocycloalkenyl containing from 5-7 ringatoms, heteroaryl or heterocyclyl containing from 5-14 ring atoms,C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl, sulfhydryl,C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy,acyl, C₂-C₂₄ alkylcarbonyl, C₆-C₂₀ arylcarbonyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, C₂-C₂₄ alkylcarbonato, C₆-C₂₀arylcarbonato, carboxy, carboxylato, carbamoyl, C₁-C₂₄ alkyl-carbamoyl,arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato,isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, C₁-C₂₄alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido, C₆-C₂₀ arylamido,sulfanamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,sulfonamide, phosphono, phosphonato, phosphinato, phospho, phosphino,polyalkyl ethers, phosphates, phosphate esters, groups incorporatingamino acids or other moieties expected to bear positive or negativecharge at physiological pH, and combinations thereof; andpharmaceutically acceptable salts thereof.
 2. The method of claim 1, theBcl-2 binding domain of IP₃R comprising the BH4 binding domain of IP₃R.3. The method of claim 1, the compound comprising a BIRD-2 (Bcl-2-IP3Rinteraction Disrupter-2) mimetic agent.
 4. The method of claim 3, theBIRD-2 mimetic agent comprising a non-peptide mimetic.
 5. The method ofclaim 1, the compound reversing the interaction of Bcl-2 with IP₃R ofcells that express IP₃R and Bcl-2.
 6. The method of claim 1, furthercomprising administering a second agent to the cells that inhibitsbinding of Bcl-2 to BH3 pro-apoptotic proteins, wherein the second agentcomprises at least one of a chromene, a thiazolidine, a benzensulfony, abenzenesulfonamide, an antimycin, an dibenzodiazocine, a terphenyl, anindole, gossypol, an apogossypol, an epigallocatechingallate, atheaflavanin,N-(4-(4-(4′-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl)-bezoyl)-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide,ABT-737, ABT-263, or ABT-199.
 7. The method of claim 6, wherein thecombination of a compound that inhibits binding of Bcl-2 to IP₃receptors (IP₃R) and a second agent that inhibits binding of Bcl-2 toBH3 pro-apoptotic proteins induces synergistic cytotoxicity of cellsthat express IP₃R and Bcl-2.
 8. The method of claim 6, the second agentcomprising at least one of a chromene, a thiazolidine, a benzensulfony,a benzenesulfonamide, an antimycin, an dibenzodiazocine, a terphenyl, anindole, gossypol, an apogossypol, an epigallocatechingallate, or atheaflavanin.
 9. The method of claim 6, the second agent comprisingN-(4-(4-(4′-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl)-bezoyl)-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide,ABT-737, ABT-263, and ABT-199.
 10. The method of claim 1, the compoundselected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 11. The method of claim1, the compound comprising the formula

and pharmaceutically acceptable salts thereof.
 12. A method of treatinga neoplastic disorder in a subject, comprising: administering to asubject's neoplastic cells expressing IP₃R and Bcl-2 a therapeuticallyeffective amount of a compound that inhibits binding of Bcl-2 to IP₃receptors (IP₃R), wherein the compound is derived from the Bcl-2 bindingdomain of IP₃R; wherein the neoplastic disorder comprises a Bcl-2associated cancer selected from the group consisting of small cell lungcancer, chronic lymphocytic leukemia (CLL), follicular lymphoma, diffuselarge B-cell lymphoma, and multiple myeloma (MM); the compound havingthe general formula (I):

wherein X¹ is CH₂, or C═O; R¹, R², R³, R⁴, and R⁵ are the same ordifferent and are independently selected from the group consisting ofhydrogen, substituted or unsubstituted C₁-C₂₄ alkyl, C₂-C₂₄ alkenyl,C₂-C₂₄ alkynyl, C₃-C₂₀ aryl, heterocycloalkenyl containing from 5-7 ringatoms, heteroaryl or heterocyclyl containing from 5-14 ring atoms,C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, halo, silyl, hydroxyl, sulfhydryl,C₁-C₂₄ alkoxy, C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₀ aryloxy,acyl, C₂-C₂₄ alkylcarbonyl, C₆-C₂₀ arylcarbonyl, acyloxy, C₂-C₂₄alkoxycarbonyl, C₆-C₂₀ aryloxycarbonyl, C₂-C₂₄ alkylcarbonato, C₆-C₂₀arylcarbonato, carboxy, carboxylato, carbamoyl, C₁-C₂₄ alkyl-carbamoyl,arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato,isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, C₁-C₂₄alkyl amino, C₅-C₂₀ aryl amino, C₂-C₂₄ alkylamido, C₆-C₂₀ arylamido,sulfanamido, imino, alkylimino, arylimino, nitro, nitroso, sulfo,sulfonato, C₁-C₂₄ alkylsulfanyl, arylsulfanyl, C₁-C₂₄ alkylsulfinyl,C₅-C₂₀ arylsulfinyl, C₁-C₂₄ alkylsulfonyl, C₅-C₂₀ arylsulfonyl,sulfonamide, phosphono, phosphonato, phosphinato, phospho, phosphino,polyalkyl ethers, phosphates, phosphate esters, groups incorporatingamino acids or other moieties expected to bear positive or negativecharge at physiological pH, and combinations thereof; andpharmaceutically acceptable salts thereof.
 13. The method of claim 12,the Bcl-2 binding domain of IP₃R comprising the BH4 binding domain ofIP₃R.
 14. The method of claim 12, the compound comprising a BIRD-2(Bcl-2-IP3R interaction Disrupter-2) mimetic agent.
 15. The method ofclaim 14, the BIRD-2 mimetic agent comprising a non-peptide mimetic. 16.The method of claim 14, the compound reversing the interaction of Bcl-2with IP₃R of neoplastic cells that express IP₃R and Bcl-2.
 17. Themethod of claim 12, further comprising administering a second agent tothe neoplastic cells that inhibits binding of Bcl-2 to BH3 pro-apoptoticproteins, wherein the second agent comprises at least one of a chromene,a thiazolidine, a benzensulfony, a benzenesulfonamide, an antimycin, andibenzodiazocine, a terphenyl, an indole, gossypol, an apogossypol, anepigallocatechingallate, a theaflavanin,N-(4-(4-(4′-chloro-biphenyl-2-ylmethyl)-piperazin-1-yl)-bezoyl)-4-(3-dimethylamino-1-phenylsulfanylmethyl-propylamino)-3-nitro-benzenesulfonamide,ABT-737, ABT-263, or ABT-199.
 18. The method of claim 17, wherein thecombination of a compound that inhibits binding of Bcl-2 to IP₃receptors (IP₃R) and a second agent that inhibits binding of Bcl-2 toBH3 pro-apoptotic proteins induces synergistic cytotoxicity ofneoplastic cells that express IP₃R and Bcl-2.
 19. The method of claim12, the compound selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 20. The method of claim12, the compound comprising the formula:

and pharmaceutically acceptable salts thereof.